The Importance of Body Fluid Identification in a Court of Law
Megan Dunkle, Cedar Crest College
Abstract: The establishment of legal systems alongside crime investigation has led to the intersection of law and science. Forensic science, at its core, is the application of scientific knowledge to questions pertaining to the law. One of the most important questions in an investigation of a crime and the proceeding court case is the identity of a suspected body fluid. Body fluid stains are found at crime scenes, and the identification of these stains is an important aspect of forensic science. Many tests currently exist to identify body fluids; however, they lack efficiency. Due to this limitation, many forensic laboratories across the country are choosing to eliminate body fluid stain identification unless requested from outside agencies. In response, other means of stain identification are under extensive research to help establish more reliable and efficient methods. With the advancement of DNA analysis, the importance of body fluid identification becomes even more crucial in a court of law. Knowing the identity of a body fluid can provide significant context to a past event and can aid in the reconstruction of the event which is always the central focus of forensic science. Without the crucial piece of stain identification, a proper reconstruction of an event cannot be achieved, even with the information provided from a DNA profile. This has been demonstrated in multiple court cases worldwide. The identification of a body fluid is an extremely important piece of information that is vital to understanding a past occurrence, especially in a court of law.
Body Fluids
Forensic science begins at the crime scene. To understand and study past events, the recognition of physical evidence is a crucial step. Physical evidence is any evidence that can provide useful information for the investigation of a crime (Lee & Pagliaro, 2013). It can be classified based on its physical state, the type of crime, and the nature of the evidence. There are multiple types of evidence that can be found at the scenes of crimes. One such type is transfer evidence which is produced through physical contact between people, objects, or people and objects. Body fluids are one of the most common types of transfer evidence encountered at crime scenes and on other pieces of evidence (Lee & Pagliaro, 2013).
The detection and identification of body fluids is an important aspect of forensic science. Blood, semen, saliva, menstrual blood, vaginal material, and skin are some of the body fluids and tissues of interest in forensic science with the first three being the ones most encountered at crime scenes (Virkler & Lednev, 2009; Harbison & Fleming, 2016). Each body fluid has a unique chemical composition and the differences between those components and/or the relative proportion of each is the primary basis for their identification (Virkler & Lednev, 2009). The identity of the body fluids can be essential for providing context to a criminal investigation and facilitating the prosecutor and/or the defense attorney in court (Harbison & Fleming, 2016).
Current Body Fluid Identification Methods
Body fluid identification was once a vital part of crime scene investigation, but with the increased use of DNA analysis, the focus has shifted, and less time has been dedicated to this important aspect of forensic science, even though many methods have been developed. Body fluid identification is still a developing area of research, with novel methods paralleling advancements in molecular biology, but time and resource limitations are causing crime laboratories to abandon body fluid identification (Forensic Scientist, Lab A & B, March 16 & 21, 2023).
There are two types of tests that are performed to identify a body fluid. The first type of tests are presumptive tests, which are simply screening tests that cannot definitively indicate the presence of a specific body fluid due to a high likelihood of false positives. There are many such tests and analysts have an extensive number of methods to choose from for most fluids. Once the presence of a body fluid is presumptively identified, a confirmatory test may be performed to conclusively indicate the presence of a body fluid. There are far fewer confirmatory tests, with only the presence of blood and semen able to be definitively confirmed (Virkler & Lednev, 2009).
Blood
Blood is the most common body fluid found at crime scenes, and many presumptive and confirmatory tests have been developed to identify it. One of the first, most important, and well-known presumptive tests is the luminol test (Virkler & Lednev, 2009; Barni et al., 2007). This test relies on the ability of several hemoglobin derivatives found in blood, to enhance the chemiluminescence of luminol when it is oxidized in an alkaline solution creating an intense blue chemiluminescence (Gaensslen, 1983). It is one of the most sensitive presumptive tests used and can even be used on areas that have been cleaned. Despite its effectiveness, there are practical limitations to its use, such as only being able to use it in dark areas (Virkler & Lednev, 2009). Another popular presumptive test is the Kastle-Meyer test. This test depends on the peroxidase activity of hemoglobin in the blood that will cause phenolphthalein in an alkaline solution to turn pink through oxidation. A huge limitation of the test is that there are many other substances, such as vegetable extracts and some body fluids (e.g., saliva), that result in a false positive (Gaensslen, 1983). While each of these presumptive tests have a value in suggesting crime scene samples may contain blood, further confirmatory analysis is needed before a definitive identification can be made.
The first commercially available confirmatory test for blood is the Rapid Stain Identification of Human Blood (RSID-Blood) which is an immunochromatographic strip test. It is designed to detect human glycophorin A, which is expressed specifically in red blood cell membranes (Independent Forensics, 2016). It utilizes the formation of an antigen-antibody-colloidal gold complex which migrates up the strip and, if glycophorin A is present, will be captured by immobile antibodies at the test and control line, resulting in a red line at both spots (Independent Forensics, 2016). Microcrystal tests are a second type of confirmatory test used to identify blood, with the most common ones being the Teichmann test and Takayama test. The Teichmann test results in the formation of the hematin derivative when blood is heated in the presence of glacial acetic acid and a halide. The resultant crystals are usually rhombic in shape and brownish in color and visible via microscopy. The Takayama test results in the formation of hemochromogen when blood is heated in the presence of pyridine and glucose under alkaline conditions. The resultant crystals are shaped like X’s and require microscopy for viewing (Greenfield et al., 2014).
Semen
The other most encountered body fluid at crime scenes is semen. Using an alternative light source (ALS), semen as well as some other body fluids, can be presumptively located and identified at a crime scene. Current ALS products on the market, however, lack either specificity or sensitivity (Santucci, 1999). The most popular presumptive test for semen identifies the presence of seminal acid phosphatase (SAP), which can catalyze the hydrolysis of organic phosphates, such as alpha-naphthyl phosphate. The product of this reaction causes a color change when combined with a diazonium salt chromogen, such as Brentamine Fast Blue (Virkler & Lednev, 2009). The test for SAP is considered presumptive due to false positives that can occur from some plant materials as well as vaginal acid phosphatase (VAP). Other issues with this test arise due to SAP degrading when exposed to heat, mold, putrefaction, or chemicals (Virkler & Lednev, 2009).
Semen can be confirmed through the microscopic identification of sperm cells. This is the most reliable and widely accepted confirmatory method for semen. Sperm cells are only found in semen and the heads contain a large amount of DNA which can be treated with a stain to make the sperm visible (Virkler & Lednev, 2009). The Christmas tree stain is the most popular stain used and gets its moniker from the resulting colors, red for the sperm heads and green for the sperm tails. A limitation of this test is that if a semen donor is azoospermic, lacking sperm in their semen, then there will be no sperm cells to identify. Quicker and easier test kits that rely on antibody-antigen reactions like RSID – Blood, have been developed (Virkler & Lednev, 2009). One such test is the One Step ABA card which utilizes a conjugation of monoclonal anti-human PSA antibodies and dye particles that can then bind human PSA. The resulting complexes migrate on the membrane of the test to a reaction and control zone. When human PSA is present, immobile antibodies at the reaction and control zones will capture the complex creating red lines. A positive test for human semen is when a red line develops at both the reaction and control zone (Hochmeister, 1999).
Saliva
Saliva is another commonly encountered body fluid on evidence. The most widely accepted presumptive tests for saliva relies on the activity of amylase (Virkler & Lednev, 2009). One of these tests is the starch-iodine test. In the presence of iodine, starch will appear blue in color. Amylase will break down the starch causing the color to change and subside. A limitation of this test is that other proteins like the albumin and gamma-globulin that are found in other body fluids will compete with the starch for the iodine, resulting in false positives (Greenfield et al., 2014). Another presumptive test relies on the activity of amylase as well as an insoluble starch-dye complex. Procion red amylopectin (PRA) reagents are used in tube tests where the amylase cleaves the starch from the dye resulting in a colored solution or in press tests where the PRA reagent is dissolved on filter paper and pressed on an item being tested to map the location of amylase-containing stains (Greenfield et al., 2014). A limitation of all these presumptive tests is that amylase is found in trace amounts in other body fluids and even in common household products, thus resulting in false positives. There are no confirmatory tests that are saliva specific, so there is no way to distinguish between true and false positive presumptive tests (Martin et al., 2006; Virkler & Lednev, 2009).
Limitations
The various current methods for body fluid identification each have their own specific challenges and problems, however, there are four big limitations that several of the methods have. The main limitation is the destructive nature of many of these tests (Virkler & Lednev, 2009). As the body fluid identification scheme is followed from presumptive tests to confirmatory tests, some of the sample is consumed for each test. If the sample is small to start, there may not be enough remaining to perform a DNA analysis. Another limitation is that all the current methods are designed to detect a specific body fluid. This means the analyst must decide which test is going to be performed, causing even more sample consumption if the results are negative. There is a need for the development of a universal test that can be applied to an unknown stain and can identify the body fluids present (Virkler & Lednev, 2009). Speaking to a couple forensic scientists across the country reveals another limitation which is the efficiency of the tests. Comparatively to DNA analysis, the body fluid identification process is much longer which results in a cost of effort and expense. The cost of the analysts’ time to perform these tests is huge. If forensic scientists are using most or all their time identifying body fluids, they are not performing DNA analysis as quickly (Forensic Scientist, Lab A & B, March 16 & 21, 2023). This can cause a serious backlog of cases which is already an issue in forensic science (Houck, 2020). These limitations are causing a shift in the field of forensic science in relation to body fluid identification.
Current Policies
Despite the multitude of current methods for body fluid identification and the prevalence of them at crime scenes, many crime laboratories in the United States are choosing to eliminate body fluid testing entirely from standard crime scene investigation. This decision is rooted in the previously discussed limitations of these methods as well as the advancements of DNA analysis. By choosing to forego body fluid stain identification, the essence of forensic science and obligations as forensic scientists to fully investigate a crime are being lost.
Forensic science is a mainstay of the criminal justice system and has a long history, however, the overall purpose of forensic science and its essence is still being redefined and debated today. A group of forensic scientists from around the world recently came together to revisit the essence of forensic science to define it and identify its fundamental principles. They determined that forensic science is a case-based, research-oriented, science-based endeavor to study the remnants of past activities through their analysis to understand unexpected events of public interest (Roux et al., 2022). These remnants or traces are the pieces of evidence collected at crime scenes and it is through their analysis that crimes can be better investigated and understood.
A key component for defining forensic science is knowing that the fundamental pieces that make up the physical record of an event are traces. Since traces are remnants, they are indicative of a former action and provide a link between the source and how it was left or the activity (Roux et al., 2022). A DNA profile found at the scene of a crime is just one example of a trace. Crime laboratories in the United States consider a DNA profile the top priority and may elect to do no other testing (Forensic Scientist, Lab A & B, March 16 & 21, 2023), however, this is failing to completely understand the DNA profile as a trace. A DNA profile on its own can provide valuable information for crime scene investigation, but to properly assign it meaning in relation to a case, the creation of the trace must be considered. One of the several important components that goes into consideration of the creation of the trace is the nature of the source (Roux et al., 2022). For a DNA profile, this is asking the question of where the DNA came from. The true answer requires more than the name of the person it originated from; it requires the specific biological material that was deposited along with the DNA. Knowing the identity of the body fluid a DNA profile originated from can help answer other questions that arise during the investigation and following court case. The DNA profile paired with its known body fluid source can provide more meaning to a case.
Traces as remnants allow for the reconstruction of past activities to better understand them. Logic and reasoning are then applied to the reconstruction to determine the actions that took place based on the information provided by traces. These findings acquire meaning in context. They do not have an intrinsic value on their own (Roux et al., 2022). A DNA profile, by itself, cannot provide all the answers necessary to help properly reconstruct an event. Crime labs across the country that choose to solely focus on obtaining DNA profiles are failing to accept the impact of context, considering body fluid identification as irrelevant information rather than recognizing it as contextual information that is necessary to provide meaning to the DNA profile.
Developing Methods
To address some of the limitations present with current body fluid stain identification methods, researchers have started exploring new techniques and methods that are based on the differential expression of genes in the cells found in each of the body fluids. The ultimate goal is to find a method that works for all body fluids. With the advancement of technology, this goal is becoming more of a reality.
RNA Typing
There are multiple types of RNA being explored to identify body fluids. Two of these kinds are messenger RNA (mRNA) and micro RNA (miRNA). The adult human body contains around two hundred distinct cell types that are specialized for each of their unique functions. The cell type is determined during gene expression with the transcription of DNA to RNA. Each specialized cell type only expresses a subset of all coding genes, and this mRNA set is what serves as the blueprint to produce proteins (Sijen, 2015). Only about two percent of the genome is translated into proteins, but at least eighty-five percent is transcribed. Various methods have been used to obtain RNA expression level information and tissue-specific mRNA profile information has been compiled in different expression portals (Hangauer et al., 2013; Lonsdale et al., 2013). miRNAs also contribute to cell differentiation and are involved with regulating mRNAs. The genes that code for miRNAs are found in introns, intergenic regions, and exons which results in them being expressed co-transcriptionally and controlled by regulatory sequences. This results in tissue-specific expression patterns (Sijen, 2015; Gomes et al., 2013). Since specialized cell types carry distinct mRNA and miRNA signatures, RNA-based methods can be used to identify body fluids found at crime scenes and on evidence.
Studies have identified multiple mRNA and miRNA markers for body fluids, allowing for identification of fluids without current identification methods. These markers may also serve as a method of organ identification which is not performed routinely in forensics (Sijen, 2015). mRNA is stable in body fluids dried on different surfaces and can be recovered with enough quality and quantity to be analyzed. miRNA is exceptionally stable postmortem and has been successfully isolated from forensically relevant samples (Harbison & Fleming, 2016). An advantage of using RNA over previously discussed protein-based methods is that it can be extracted simultaneously with the DNA used for profiling (Sijen, 2015). Some countries have begun integrating RNA typing in their forensic casework, and it has been used in the courtroom as well. This shows the ultimate value of RNA typing and that it can be applied to not only forensic casework, but also has held up in court and provided valuable information (Sijen & Harbison, 2021).
DNA Methylation
DNA methylation occurs at the 5’-position of cytosine in a dinucleotide pattern of 5’-CG-3’. These sites are known as CpG sites and are genetically programmed DNA modifications in mammals. These markers are important for gene expression in eukaryotic organisms and thus the differentiation of cells and their subsequent development into tissues, organs, and organ systems (Ohgane et al., 2008). The pattern of DNA methylation may change during early in utero development, however, once cellular differentiation occurs, these patterns become more established. DNA methylation patterns in differentiated cells show a limited dynamic range in normal conditions (Lee et al., 2012). This gives various cells and tissues a specific methylation profile of tissue-dependent differentially methylated regions and these profiles provide a way to distinguish between cell and tissue types (Ohgane et al., 2008). Due to this distinguishability between cell types, DNA methylation analysis is an emerging technique to identify body fluids in forensic science.
Recent studies have found multiple CpG markers in various body fluids that only produce a methylation signal in the target body fluid (Park et al., 2014). This also allows for the identification of body fluids that currently do not have other identification methods. These markers have been applied to actual forensic casework and demonstrated that DNA methylation analysis can be used in a real-world setting. DNA methylation analysis also works on older cases, providing an invaluable tool for when body fluids could not be identified at the time of the crime, but can now be analyzed to identify the DNA source through DNA methylation assays (Choung et al., 2021). An advantage of DNA methylation is that there is only one extraction required for both methylation and DNA profiling analysis, which allows for an easier process and shorter preparation time. When using RNA methods, DNA and RNA must be extracted simultaneously, providing an issue when there is a limited amount of evidence. The analysis of RNA uses complex methods and is more unstable than DNA, therefore methods utilizing RNA are not as practical in areas of the world that have mandated timelines of when results are needed (Choung et al., 2021). The application of DNA methylation analysis to actual forensic casework and its compatible methods shows the potential of its implementation into forensic labs across the country.
The Courtroom
Understanding what occurred during a past event is a crucial part of a lawyer’s role in the courtroom. Crime scene reconstruction is taking the various pieces of evidence and developing a scenario of what could have occurred before, during, and after the commission of a crime. In a courtroom, the prosecution and the defense will both present different scenarios (Sijen & Harbison, 2021). These hypotheses need to be well thought out, describe the supposed events, and be mutually exclusive. Body fluid identification provides a critical link between the donor and the activities that occurred. This link is important because it adds evidence to the investigation, and subsequent court case (Sijen & Harbison, 2021).
Most of the cases that come to court involving any cellular material do not revolve around the dispute of the presence of it, but rather what caused the deposition of such material. The central focus is how that individual’s DNA got to the crime scene. These questions are approached through activity-level evaluations by forensic scientists (Sijen & Harbison, 2021). When considering activity-level evaluations, three fundamental principles are considered. The findings should be assessed within a framework of circumstances, the probability of the findings based on the propositions is assessed, not the probability of the propositions themselves, and the findings should be evaluated against two competing, mutually exclusive scenarios (Taylor et al., 2018). The activity-level evaluations weigh the likelihood of one scenario over the other. These different scenarios are known as the prosecution and defense scenarios (Sijen & Harbison, 2021).
The analysis of the evidence is used to relate the cellular material that either matches the victim or suspect to one of the following scenarios. Under the prosecution scenario, the suspect is the offender and the evidence found and analyzed is indicative of an offensive activity (Sijen & Harbison, 2021; Gill, 2014). For example, blood on the blade of the knife matching the victim and cellular material on the handle matching the suspect supports the scenario that the suspect conducted the stabbing. Under the defense scenario, a different, unknown person is the offender and there are alternative scenarios that explain the findings related to the analysis of the evidence (Sijen & Harbison, 2021; Gill, 2014). Using the previous stabbing example, an alternative scenario would be that the suspect’s cellular material is the result of secondary transfer. Being able to identify the source of a DNA profile is critical to address the various hypotheses detailed in a courtroom (Sijen & Harbison, 2021).
Identifying the presence of body fluids at crime scenes and on evidence, as well as determining the source of a DNA profile, can significantly impact a case. Body fluid identification is just one vital piece in understanding and contextualizing the scene. Without knowing what body fluids are present, then the location, pattern, and amount of cellular material become obsolete (Sijen & Harbison, 2021). This information then would not be available to assess the different scenarios presented. A crucial link between the evidence and the alleged crime can be provided through body fluid identification. It can also provide more context to what occurred during the commission of a crime, which is then used to confirm or deny the differing scenarios presented in court (Sijen & Harbison, 2021). In the instance of secondary transfer, knowing what body fluids are present allows analysts to assess the likelihood of those body fluids being present at the primary location (Bouzga et al., 2020). Without body fluid identification, a crime might not be fully or falsely contextualized which would be detrimental in a courtroom.
When it comes to presenting a case in court, understanding the different scenarios that could have occurred during a past event is critical for a lawyer. Body fluid identification is an important piece of an investigation that helps reconstruct a past event (Sijen & Harbison, 2021). It provides a link between the suspect, victim, or both to the alleged crime as well as provides context for the evidence and what activities might have occurred. Knowing what body fluids are present at a crime scene supplies significant information and has the potential to influence a court case (Sijen & Harbison, 2021).
Case Studies
Neill-Fraser v Tasmania (Neill-Fraser v Tasmania, 2021)
On January 27, 2009, some locals noticed a yacht sitting lower in the water than expected and notified the police department. When the police boarded the yacht, they found signs of a violent altercation. There were reddish brown stains located around the yacht as well as a knife. Robert Chappell, one of the owners of the yacht could not be found, however, a DNA profile obtained from one of the stains was consistent with his profile.
Deborah McHoul, forensic scientist, used luminol to presumptively identify blood on the yacht. One of these stains provided a DNA profile matching an unknown female. In March 2010, this profile was found to be consistent with a young girl from the area. Due to luminol’s sensitivity and limitations, it was possible that there was a tiny amount of blood mixed in with some other body fluid and the DNA’s source could not be concluded as blood. The girl stated that she had never been on the yacht nor been to the other areas where it had been docked but would go on to change her statement various times in the following years.
Despite Chappell’s body never being recovered, the victim’s wife was found guilty of killing her husband at the original trial in October 2010. She tried to appeal her case twice, most recently in 2021. Both times, the defense relied heavily upon the discovery of the girl’s DNA. Carl Grosser and Maxwell Jones, two forensic scientists, each testified at one of the appeals. Grosser testified that a person’s DNA profile could be deposited through secondary transfer if, for example, someone had stepped into a body fluid and transferred it to the deck on their shoe. While he had never worked on a case where this occurred, he testified that it was theoretically possible, however, he could not make any assessment about the possibility of transfer due to not knowing the DNA’s source. Both Jones and Grosser concluded that based on the electropherogram, the girl’s DNA profile was a strong one, with characteristics inconsistent with a contact DNA scenario. Due to its strength, they also concluded that her DNA profile was indicative of a large amount of DNA which was more likely to come from a body fluid than a touch event. Using Grosser’s example of transfer, Jones went on to say that another stain resulting in the same DNA profile would have been expected to be deposited as the person moved about the yacht. Since no similar stain was located, he concluded that there was nothing to support the hypothesis of secondary transfer due to foot traffic.
The circumstantial evidence indicating the wife’s guilt was compelling, however, the presence of the girl’s DNA profile was not considered strongly, and the idea of secondary transfer was highly regarded. Had more body fluid identification methods been used, a more accurate conclusion of the source of the girl’s DNA profile and her involvement could have been obtained. This could have altered the outcome of the original trial or the following appeals.
Case Number 20-002409-13 (2014)
In February of 2012, the victim was found dead in his car with multiple gunshots wounds to his head and upper body area. During the initial search of the vehicle, investigators noted snow on the bottom of the victim’s shoes and on the passenger’s side floor. Where the victim’s car had been found, it had recently snowed, and they concluded that the victim and another person had gotten in the victim’s car after walking through the snow. Through the victim’s cell phone data, they established that he had called the suspect several times the night he died, and cell towers placed the suspect in the area where the victim’s car was found. Investigators seized a jacket worn by the suspect and analyzed it for gunshot residue and body fluids. They confirmed the presence of gunshot residue as well as traces of blood. A DNA profile generated from the blood was consistent with the victim. The suspect was eventually charged and went to court. During the trial, the defense tried to argue that if the suspect had been the shooter, then more blood would have been found on the sleeves of his jacket. Further testing using RNA typing was conducted on the traces of blood. Scientists were able to confirm not only the presence of blood, but skin cells and brain tissue as well. The blood, skin cells, and brain tissue all had a DNA profile consistent with the victim. The identification of the brain tissue was able to disprove the defense’s scenario and the suspect was eventually charged with manslaughter. Tissue typing specifically played a vital role in the prosecution of this case.
Conclusion
Body fluids are some of the most common types of transfer evidence found at crime scenes and on other pieces of evidence (Lee & Pagliaro, 2013). This makes their detection and identification a crucial aspect of crime scene investigation and forensic science. Currently, there are many methods for the identification of body fluids, however, these methods have severe limitations. Due to these limitations, body fluid identification is being considered less important than other investigative leads like DNA analysis, and crime laboratories in the United States are choosing to forgo these assays (Forensic Scientist, Lab A & B, March 16 & 21, 2023) and failing in their obligations to fully investigate a crime. To alleviate the limitations associated with current testing methods, researchers are developing new methods. This research is incredibly important because conserving body fluid identification as a critical part of standard crime scene investigation procedures will help assist the investigation and subsequent court case. Knowing the identity of a body fluid and thus the source of a DNA profile, allows the jury to better contextualize and understand the case. It can also provide information that is important for confirming or denying scenarios presented in court for how the evidence was deposited or what had occurred at the scene of a crime. In numerous cases, the identity of a body fluid or tissue has played a significant role in the prosecution of court cases. Alternatively, when such testing was omitted, the outcome of the court case suffered from lack of information. This demonstrates how vital and important body fluid identification is to crime scene investigation, legal proceedings, and forensic science as a whole.
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